The catecholamines dopamine, norepinephrine, and epinephrine are the principal neurotransmitters in the sympathetic nervous system. In particular, norepinephrine regulates many critical functions that include attention, memory, learning, emotion, and autonomic and cardiovascular function. In the periphery, norepinephrine increases heart rate, cardiac contractility, vascular tone, renin-angiotensin system activity, and renal sodium reabsorption. Norepinephrine is secreted by chromaffin cells, which package catecholamines at high concentrations (0.5-1.0 M) and at low pH (5.0-5.5) in neurosecretory vesicles. Chromaffin cells possess approximately 30,000 large dense-core vesicles (LDCV) with norepinephrine and epinephrine. Chromaffin cells that store and release mainly epinephrine can be separated from those that utilize mainly norepinephrine through density-gradient centrifugation, though a third subpopulation which secrete both epinephrine and norepinephrine has been identified via cyclic voltametry. Over the years, chromaffin cells have become a standard platform for the study of processes related to exocytosis. Thus, chromaffin cells appeared to be an ideal platform for the study of novel sensors for neurotransmitters.
Currently, catecholamines can be studied via electrochemical and chromatographic techniques that provide characterization and quantification, although these techniques can only provide crude spatial information. Recently, fluorescent false neurotransmitters (FFNs) have been developed which are selectively loaded into vesicles that express neuronal vesicular monoamine transporter (VMAT) and represent an optical approach for labeling vesicles containing catecholamines and imaging catecholamine release at the single-vesicle level. However, FFNs are loaded into all secretory vesicles expressing the VMAT protein without discrimination to cell type and thus, the approach cannot distinguish distinct cell populations that secrete a particular neurotransmitter.
Fluorescent sensors remain a compelling technology for approaching the general problem of selective neurotransmitter detection. In recent years, a number of catecholamine sensors have been reported including RNA aptamers, fluorescent ribonucleopeptide (RNP) complexes, and boronic-acid based synthetic receptors. However, none of these methods represent a practical approach for in vivo and in vitro cellular analysis and imaging. Indeed, some time ago, the inventors developed a coumarin aldehyde fluorescent sensor,
disclosed in U.S. Pat. No. 7,977,120 B2, for the selective recognition and sensing of amines. The '120 sensor, along with its derivatives, includes an aldehyde that associates with the analyte amine group via imine formation and a boronic acid that associates with the catechol group. Unfortunately, the catechol group strongly quenched the sensor. Thus, in the case of dopamine and norepinephrine, the sensor operated in a turn-off mode.
Therefore, there is a need for a series of fluorescence chemosensors to detect an organic primary-amine-containing analyte, such as, neurotransmitters and diamino-analytes, in a live cell. There is also a need for a series of fluorescence chemo sensors to operate in a turn-on mode when detecting dopamine and norepinephrine.